Measuring multiple parameters of CD8+ tumor-infiltrating lymphocytes in human cancers by image analysis

Abstract

BackgroundImmuno-oncology and cancer immunotherapies are areas of intense research. The numbers and locations of CD8+ tumor-infiltrating lymphocytes (TILs) are important measures of the immune response to cancer with prognostic, pharmacodynamic, and predictive potential. We describe the development, validation, and application of advanced image analysis methods to characterize multiple immunohistochemistry-derived CD8 parameters in clinical and nonclinical tumor tissues.MethodsCommercial resection tumors from nine cancer types, and paired screening/on-drug biopsies of non–small-cell lung carcinoma (NSCLC) patients enrolled in a phase 1/2 clinical trial investigating the PD-L1 antibody therapy durvalumab (NCT01693562), were immunostained for CD8. Additional NCT01693562 samples were immunostained with a CD8/PD-L1 dual immunohistochemistry assay. Whole-slide scanning was performed, tumor regions were annotated by a pathologist, and images were analyzed with customized algorithms using Definiens Developer XD software. Validation of image analysis data used cell-by-cell comparison to pathologist scoring across a range of CD8+ TIL densities of all nine cancers, relying primarily on 95% confidence in having at least moderate agreement regarding Lin concordance correlation coefficient (CCC = 0.88–0.99, CCC_lower = 0.65–0.96).ResultsWe found substantial variability in CD8+ TILs between individual patients and across the nine types of human cancer. Diffuse large B-cell lymphoma had several-fold more CD8+ TILs than some other cancers. TIL densities were significantly higher in the invasive margin versus tumor center for carcinomas of head and neck, kidney and pancreas, and NSCLC; the reverse was true only for prostate cancer. In paired patient biopsies, there were significantly increased CD8+ TILs 6 weeks after onset of durvalumab therapy (mean of 365 cells/mm2 over baseline; P = 0.009), consistent with immune activation. Image analysis accurately enumerated CD8+ TILs in PD-L1+ regions of lung tumors using the dual assay and also measured elongate CD8+ lymphocytes which constituted a fraction of overall TILs.ConclusionsValidated image analysis accurately enumerates CD8+ TILs, permitting comparisons of CD8 parameters among tumor regions, individual patients, and cancer types. It also enables the more complex digital solutions needed to better understand cancer immunity, like analysis of multiplex immunohistochemistry and spatial evaluation of the various components comprising the tumor microenvironment.Trial registrationClinicalTrials.gov identifier: NCT01693562.Study code: CD-ON-MEDI4736–1108.Interventional study (ongoing but not currently recruiting).Actual study start date: August 29, 2012.Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0326-x) contains supplementary material, which is available to authorized users.

Prevalence of CD8+ cytotoxic lymphocytes in human neoplasms

“…Another study that counted T cells manually in 102 cervical squamous cell cancers reported a median of 441 CD8 + T cells/mm 2 , which is very similar to the median value of 468 CD8 + T cells/mm 2 in our 43 patients [20]. One earlier study by Steele et al [28] has used "cells/mm 2 " to compare the density of CD8 + cells in 8 different cancer types in a study analyzing the largest possible area per cancer on large sections. Several relevant observations were comparable between our and their study (Table 2), such as relatively low densities of CD8 + cells in kidney and prostate cancers and a high density in non-small cell lung cancer.…”

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“…The deficiency of CD8+ T cells have been shown in experimental models to result in tumor progression, while tumor regression was observed in the CD8+ proficient model [ 27 ]. The spatial distribution of CD8+ T cells within the tumor microenvironment were very variable among different patients and different cancer types [ 28 ]. Those distribution of CD8+ T cells were likely related with prognosis, with dense CD8+ T cells between the tumor being more positive than dense tumor stromal CD8+ T cells [ 29 ].…”